Purification and characterization of an exo-beta-1,4-glucanase from Ruminococcus flavefaciens FD-1

Abstract

An exo-0-1,4-glucanase (Exo A) from Ruminococcus flavefaciens FD-1 was purified to homogeneity and characterized. Enzyme activity was monitored during purification by using the substrate p-nitrophenyl-o-Dcellobioside (NPC). Over 85% of the NPC activity was found to be extracellular once the filter paper was degraded (7 days). Culture supernatant was harvested, and the protein was concentrated by ultrafiltration. The retentate (.300,000 Mr), containing most of the activity against NPC, was then fractionated with a TSK DEAE-5PW column. This yielded a sharp major peak of NPC enzyme activity, followed by a broader, less active area that appeared to contain at least six minor peaks of lower enzymatic activity. Further purification was achieved by chromatography with a hydroxylapatite column. Finally, gel ifitration chromatography yielded a homogeneous enzyme (Exo A) as determined by silver stains of both sodium dodecyl sulfate-and nondenaturing electrophoresis gels. Substrate specificity experiments and the products of cellulose digestion indicate that the enzyme was an exo-,3-1,4-glucanase. Exo Cellulolytic organisms produce several cellulase enzymes which have different specificities and modes of action. At least three types of cellulase enzymes are involved in the degradation of cellulose by fungi (25). Endo-P-1,4-glucanase (EC 3.2.1.4) is produced by all cellulolytic microorganisms and is more commonly referred to as carboxymethylcellulase (CMCase) or C, cellulase. Exo-P-1,4-glucanase (EC 3.2.1.91) is also referred to as C1 cellulase or cellobiohydrolase. ,-1,4-Glucosidase (EC 3.2.1.21) is widely distributed among organisms and is commonly known as cellobiase or P-glucosidase. The most efficient and complete hydrolysis of cellulose is thought to be the result of the combined synergistic action of both endoglucanase and exoglucanase.Two of the most important cellulolytic bacteria in the rumen are Ruminococcus flavefaciens and Ruminococcus albus (2, 11). Typically, these two species ferment cellulose, cellobiose, and xylan. The cellulase systems of both R. flavefaciens and R. albus appear to have many similarities. However, in general, R. flavefaciens degrades crystalline cellulose more efficiently than R. albus (2, 11). This observation suggests that R. flavefaciens possesses exoglucanase activity.Pettipher and Latham (29) investigated the cellulase complex from R. flavefaciens as a crude preparation and concluded that the most active enzymes present in the complex were of the exo-,-1,4-glucanase type. However, the authors did not purify any of the enzymes present or investigate the number and types of cellulolytic enzymes in the different molecular weight complexes. The authors concluded that the major enzyme type was exo-P-1,4-glucanase, based on the products of cellulolytic activity (cellobiose, cellotriose, and a small amount of glucose) and the changes in viscosity versus reducing sugar. However, valid interpretations of cellulase enzyme type cannot be made from data generated * Corresponding author. by a m...

Isolation and properties of a major cellobiohydrolase from the cellulosome of Clostridium thermocellum

“…The levels of cellobiose required for halfmaximal inhibition on various substrates (0.05 to 0.1%) are similar to that observed for a cellobiohydrolase derived from Ruminococcusflavefaciens (11). Similar levels of cellobioseinduced inhibition have been described for both the purified cellulosome (22,23) and the crude cellulase system (15) of C. thermocellum.…”

“…It should also be mentioned in this context that stabilization by Ca2" ions was recently described for another known cellulosomal subunit, i.e., cloned EGD (corresponding to the S11 subunit), expressed in E. coli (6). In addition, one other confirmed cellobiohydrolase (from R. flavefaciens) has also been shown to require Ca2" for maximal activity (11).…”

Characterization of a Cellobiohydrolase (MoCel6A) Produced by Magnaporthe oryzae

“…Competitive inhibition by end products is a significant issue for not only cellobiohydrolases, but also other hydrolytic enzymes in efforts to establish conditions for the efficient degradation of carbohydrates. Cellobiose is the main product liberated from cellulose derivatives by the action of cellobiohydrolases and typically decreases the hydrolytic activity of cellobiohydrolases (13,15,28,31,36,37,44). The activity of T.…”

“…Also, Trichoderma reesei Cel6A can hydrolyze 1,3-1,4-␤-glucan (1,18), but it is unclear whether in vivo it is hydrolysis of 1,3-1,4-␤-glucan that occurs mainly or hydrolysis of cellulose derivatives. The resulting accumulation of cellobiose inhibits the activity of cellobiohydrolase (13,15,28,31,36,37,44). Some microorganisms possess cellulosomes, multienzyme complexes that contribute to the efficient degradation of cellulose.…”

Cellulose and Xylan Degrading Enzymes of the Plant Pathogenic Fungus,Fusarium oxysporumSUF850

“…One unit of CMCase and xylanase activity was defined as the amount of enzyme releasing II/mol of reducing sugar per minute at 40°C. The activity of p-nitrophenyl-fi-D-cellobiosidase (pNPCase) was assayed by the method of Gardner et a1 9 ) One unit of pNPCase was defined as the amount of enzyme releasing II/mol of p-nitrophenol at 40 D C per minute. The activity as to the reduction of the viscosity of a CMC solution was determined for the selection of strains producing higher levels of cellulolytic enzymes.…”